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Agonistic Properties of Cannabidiol at 5-HT1a Receptors

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Cannabidiol (CBD) is a major, biologically active, but psycho-inactive component of cannabis. In this cell culture-based report, CBD is shown to displace the agonist, [3H]8-OH-DPAT from the cloned human 5-HT1a receptor in a concentration-dependent manner. In contrast, the major psychoactive component of cannabis, tetrahydrocannabinol (THC) does not displace agonist from the receptor in the same micromolar concentration range. In signal transduction studies, CBD acts as an agonist at the human 5-HT1a receptor as demonstrated in two related approaches. First, CBD increases [35S]GTPgammaS binding in this G protein coupled receptor system, as does the known agonist serotonin. Second, in this GPCR system, that is negatively coupled to cAMP production, both CBD and 5-HT decrease cAMP concentration at similar apparent levels of receptor occupancy, based upon displacement data. Preliminary comparative data is also presented from the cloned rat 5-HT2a receptor suggesting that CBD is active, but less so, relative to the human 5-HT1a receptor, in binding analyses. Overall, these studies demonstrate that CBD is a modest affinity agonist at the human 5-HT1a receptor. Additional work is required to compare CBD's potential at other serotonin receptors and in other species. Finally, the results indicate that cannabidiol may have interesting and useful potential beyond the realm of cannabinoid receptors.
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Agonistic Properties of Cannabidiol at 5-HT1a Receptors
Ethan B. Russo,
1
Andrea Burnett,
1
Brian Hall,
1
and Keith K. Parker
1,2
(Accepted June 27, 2005)
Cannabidiol (CBD) is a major, biologically active, but psycho-inactive component of
cannabis. In this cell culture-based report, CBD is shown to displace the agonist, [3H]8-OH-
DPAT from the cloned human 5-HT1a receptor in a concentration-dependent manner. In
contrast, the major psychoactive component of cannabis, tetrahydrocannabinol (THC) does
not displace agonist from the receptor in the same micromolar concentration range. In signal
transduction studies, CBD acts as an agonist at the human 5-HT1a receptor as demonstrated
in two related approaches. First, CBD increases [35S]GTPcS binding in this G protein coupled
receptor system, as does the known agonist serotonin. Second, in this GPCR system, that is
negatively coupled to cAMP production, both CBD and 5-HT decrease cAMP concentration
at similar apparent levels of receptor occupancy, based upon displacement data. Preliminary
comparative data is also presented from the cloned rat 5-HT2a receptor suggesting that CBD
is active, but less so, relative to the human 5-HT1a receptor, in binding analyses. Overall, these
studies demonstrate that CBD is a modest affinity agonist at the human 5-HT1a receptor.
Additional work is required to compare CBD’s potential at other serotonin receptors and in
other species. Finally, the results indicate that cannabidiol may have interesting and useful
potential beyond the realm of cannabinoid receptors.
KEY WORDS: Cannabis; cannabidiol; cAMP; G Proteins; marijuana; serotonin; THC.
INTRODUCTION
Although cannabis and its extracts have been
extensively studied, knowledge of the biochemical
mechanisms of one of its major components, canna-
bidiol (CBD), has not been thoroughly explored (1,2).
This lack of knowledge of CBD’s biochemical phar-
macology is noteworthy in the context of its known
potential in human therapy: for example, it has been
demonstrated to have anxiolytic (3), anti-seizure (4),
anti-psychotic (3), and neuroprotective properties
(5,6). While previously thought to be sedating, recent
clinical research has confirmed that CBD is activat-
ing, and that it counters sedative effects of THC (7).
The major psychoactive component of cannabis,
tetrahydrocannabinol (THC), has received extensive
research attention into its biochemical pharmacology.
Both THC and CBD have been pharmacologically
investigated at cannabinoid receptors (CBR), which
are highly conserved across animal taxa, with the
major exception of insects (8–10). THC is at least 10
times more potent in binding to CB1 receptors than
CB2 receptors. At CB1R, there is evidence to suggest
that CBD is an antagonist or inverse agonist, al-
though substantial debate still exits about its intrinsic
activity (10,11). CBD has received little attention in
other neurotransmitter systems. Noteworthy in this
regard is serotonin (5-hydroxytryptamine; 5-HT),
which is known to be involved in many of the same
processes important to cannabis’s actions (12,13) such
as relief of anxiety, pain, the complex processes of
1
Skaggs School of Pharmacy, The University of Montana,
Missoula, MT 59812-1552, USA.
2
Address reprint requests to: Keith K. Parker, Skaggs School of
Pharmacy, The University of Montana, Missoula, MT 59812-
1552, USA. Tel.: +406-243-4235; Fax: +406-243-5228; E-mail:
keith.parker@umontana.edu
Neurochemical Research, Vol. 30, No. 8, August 2005 (Ó 2005), pp. 1037–1043
DOI: 10.1007/s11064-005-6978-1
1037
0364-3190/05/0800–1037/0 Ó 2005 Springer Science+Business Media, Inc.
headache (14,15), and thermoregulation. The few
studies done with CBD in serotonergic systems
suggest that it inhibits 5-HT re-uptake, and overall
reduces 5-HT neurotransmission (2,16). There is also
some experimental evidence to support CBD’s activity
in other neurotransmitter systems such as dopamine,
GABA, and the endogenous opioid system (2).
Most of 5-HT’s broad actions are thought to be
regulated at a series of 5-HT receptors (5-HTR), the
majority of which (17) are members of the diverse
super family of G-protein coupled (GPC), seven-
transmembrane receptors (7TMR). The 5-HT1aR
(17) has been cloned and studied in numerous in vivo
and cell culture systems and in various species. It has
been cloned in both human (H) and rat (18–20),
amongst other organisms, and has been further
analyzed in other species, including rabbit (21), where
it has not been cloned. In this literature, extending
over two decades, 5-HT1aR has been ever more
implicated in a variety of physiological and
pathological processes including anxiety, mood,
depression, panic, obsessive-compulsive disorders,
headache, immune regulation, and cardiovascular
regulation to name a few (2,6,17,18). Additionally, the
5-HT2aR could have relevance to the pharmacology
of cannabis as it has been associated with phenomena
like mood, headache, and hallucination (22). There is
precedence for the action of cannabinoids such as
oleamide at serotonin receptors (23–26).
Over the last decade our laboratory has con-
ducted a series of studies with 5-HT1aR (27), and to a
lesser extent with 5-HT2aR (21). Because of these
interests and our hypothesis that CBD may have
important actions relevant to the pharmacology of
cannabis but outside the realm of CBR, we report
here studies with H5-HT1aR a nd a limited compar-
ison to the rat 5-HT2aR (28). For both H5-HT1aR
and rat 5-HT2aR we also report comparisons be-
tween CBD and THC. In cell culture experiments
with cloned human 5-HT1aR and rat 5-HT2aR,
CBD has a greater affinity than THC for both
receptors. CBD binds with higher affinity at 5-
HT1aR than at 5-HT2aR. In the case of H5-HT1aR,
CBD appears to act as an agonist. A preliminary
report of these investigations has appeared (29).
EXPERIMENTAL PROCEDURE
Cell Culture. Chinese Hamster Ovary (CHO) cells expressing
the H5-HT1aR (19) were cultured in Ham’s F-12 medium fortified
with 10 % fetal calf serum and 200 ug/ml geneticin. Cultures were
maintained at 37°C in a humidified atmosphere of 5% CO2. Cells
were sub-cultured or assayed upon confluency (5–8 days). Cloned
H5-HT1aR was kindly provided by Dr. John Raymond (Medical
U. of South Carolina). NIH 3T3 cells expressing the rat 5-HT2aR
(28) were cultured under similar conditions in DMEM fortified
with 10% calf serum and 200 lg/ml geneticin. These transfected
cells were generously provided by Dr. David Julius (UCSF). Both
cell lines have been tested for mycoplasma with a PCR kit (ATCC),
and are free of contamination.
Receptor Preparation. Cells were harvested by trypsinization
and centrifuged at low speed in ice-cold medium. The pellet was re-
suspended in ice-cold Earle’s Balanced Salt Solution followed by
centrifugation. Cells were re-suspended in 10 ml of ice-cold binding
buffer (50 mM Tris, 4 mM CaCl2, 10 lM pargyline, pH 7.4),
homogenized with Teflon-glass, and centrifuged for 450,000 g-min.
at 4°C. To produce a crude membrane preparation, the pellet was
re-suspended in 30 ml of ice-cold binding buffer, and homogenized,
first with Teflon-glass and then with a Polytron (setting 4) for 5 s.
The receptor preparation was stored on ice and assayed within the
next 1.5 h.
Assay of Receptor Activity. Binding of the agonist [3H]8-OH-
DPAT ([3H]8-hydroxy-2-(di-n-propylamino)tetralin) to H5-
HT1aR followed well-characterized in vitro protocols (20,27,30).
Radioligands were purchased from New England Nuclear (NEN),
Boston, MA. 1 ml reaction mixtures, in triplicate, were incubated
for 30 min. in a 30°C shaker bath. Composition of the 1 ml reac-
tion mixture was: 700 ll of receptor preparation; 100 ll of either
binding buffer (for total binding) or 10 lM 5-HT (final concen-
tration for non-specific binding), 100 ll of the tritiated agent (final
concentration of 0.5 nM [3H] 8-OH-DPAT), and 100 ll of diluted
CBD or binding buffer in the case of controls.
Reactions were stopped by addition of 4 ml of ice-cold
50 mM Tris buffer, pH 7.4, and subsequent vacuum filtration on
glass fiber filters (Whatman GF/B). Filters were rinsed twice in
5 ml of ice-cold Tris buffer, dried, and counted in 5 ml of Ecoscint
(National Diagnostics) liquid scintillation fluid in a Beckman LS
6500 instrument. Homogenates were assayed for protein to main-
tain a nominal value of 50 lg protein per filter over weekly assays
(31). Total and non-specific binding tubes were run in triplicate.
Assays of the rat 5-HT2aR (28) were conducted under similar
conditions with the 1 ml reaction mixture containing: 700 llof
receptor preparation; 100 ul of either binding buffer (for total
binding) or 10 lM mianserin (final concentration for non-specific
binding); 100 ll of the tritiated agent (final concentration of
0.2 nM [3H] ketanserin); and 100 ll of diluted CBD or binding
buffer in case of controls.
cAMP Assay. CHO cells were cultured to confluency in 12-
or 24-well plates (27). Medium was aspirated and the cells were
rinsed twice in warm, serum-free F-12 medium. Cells were then
incubated for 20 min. at 37°C in 0.5 mls of serum-free F-12 med-
ium containing 100 lM isobutylmethylxanthine (IBMX) and the
following substances (final concentrations) alone or in combination
(see Fig. 3): 30 lM forskolin (FSK; for all treatments); 1 lM5-
HT; 16 lM CBD; and 0.05 lM NAN-190 (NAN). Reactions were
stopped by aspiration of medium and addition of 0.5 ml of
100 mM HCl. After 10 min., well contents were removed and
centrifuged at 4000 rpm. Supernatants were diluted in 100 mM
HCl, and cAMP was quantified (27) directly in a microplate format
by colorimetric enzyme immunoassay (EIA) with a kit from Assay
Designs (Ann Arbor). Triplicate independent samples were assayed
in quadruplicate to increase precision.
[35S]GTPcS Assay. H5-HT1aR membranes from trans-
fected CHO cells were incubated with 5-HT (0.1 lM) and/or CBD
1038 Russo, Burnett, Hall, and Parker
(16 lM); see Fig.2), and the following incubation mixture: 20 mM
HEPES buffer, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT,
100 mM NaCl, 100 uM GDP, 10 lM pargyline, 0.2 mM ascor-
bate, and 0.1 nM [35S]GTPcS (32). Mixtures were incubated for
30 min. at 30°C, and were terminated by dilution in cold buffer.
The mixture was filtered on GF/C filters, rinsed twice in buffer,
followed by drying and liquid scintillation counting. Negative
control (basal incorporation) was the above mixture minus CBD or
5-HT. Non-specific binding was determined in the presence of cold
GTPcS-(10 lM). Positive control was H5-HT1aR membranes in
the same incubation mixture plus 5-HT. All values reported in
Fig. 2 are for specific binding (total non-specific) of triplicates.
Dilution of Cannabinoids. CBD and THC were obtained in
dilute (1 mg/ml) solution from Sigma Chemical Co. (St. Louis,
MO). These solutions were stored at 4°C until use and then diluted
in distilled water and finally in the buffer appropriate to the par-
ticular assay. Fresh dilutions of cannabinoids were made daily.
Each final concentration of cannabinoid thus contained some of
the vehicle (methanol). The highest concentration of methanol
encountered in any assay (1%) was then tested in that assay system
for activity. In pair-wise comparative t testing, none of the meth-
anol controls were found to be distinguishable from negative
control (buffer).
Statistical Analysis. All statistics (means, standard devia-
tions, standard errors of the mean (SEM), and t tests) were per-
formed with software provided by Poly Software International; in
some cases, statistics were corroborated by hand using a Hewlett-
Packard Graphing Calculator, HP48. Graphs were constructed
with Excel software provided by Microsoft.
RESULTS
Cannabidiol produces concentration-dependent
displacement of the agonist [3H[8-OH-DPAT from
the H5-HT1aR (Fig. 1). Using crude membrane
preparations from cultured CHO cells transfected
with H5-HT1aR (See methods), CBD diluted in
methanolic buffer displaced agonist by 73 ± 8 %
(S.E.M.) at 16 lM. The highest concentration of
methanol (1%) present at 32 lM CBD produced
only 3 ± 0.5% displacement of agonist, a level
indistinguishable from control when the methanol
and control means are compared statistically. While
CBD was active in the micromolar range, tetrahy-
drocannabinol was unable (108 ± 6% of control) to
produce agonist displacement at a concentration of
32 lM.
The ability of CBD to produce concentration-
dependent displacement of highly potent and specific
agonist from the H5-HT1aR ligand-binding site
raised the question of the intrinsic activity of CBD.
Experiments were designed to test the agonistic
potential of cannabidiol. Since H5-HT1aR is G pro-
tein-coupled, agonist binding would be expected to
increase GTP binding, measurable when the stable
analog of GTP, GTP cS is present in a radiolabeled
form. 0.1 lM 5HT increased [35S]GTPcS incorpora-
tion by 57 ± 7% (Fig. 2) above the basal level (buffer)
in membranes of CHO transfected with the receptor.
Similarly, 16 lM CBD increased [35S]GTPcS incor-
poration by 67 ± 6% above the basal level. Together,
5-HT and CBD increased [35S]GTPcS incorporation
to 123 ± 10% above the basal level, suggesting that
CBD had not reached its maximum possible stimu-
Fig. 1. Displacement of Specifically-Bound [3H]8-OH-DPAT By Cannabidiol (CBD) and Tetrahydrocannabinol (THC) In Membranes
Containing the Human 5-HT1a Receptor. Concentrations are micromolar. Values are the mean ± SEM with n’s=3–6. More detailed
experimental conditions of cell culture, membrane preparation, and drug-receptor binding are outlined in Experimental Procedure.
5-HT1a Receptor Agon ism by Cannabidiol 1039
lation. By reference to CBD’s displacement capacity
at the receptor’s ligand binding site (Fig. 1), 16 lM
CBD occupies about 73% of the available binding
sites.
To further test the hypothesis that CBD is an
agonist at H5-HT1aR, experiments were designed to
measure activity in the adenylyl cyclase (AC) system
negatively coupled to the receptor. In this format, AC
is first stimulated by the natural product forskolin
(FSK) at a concentration of 30 lM (control = 100 ±
5%). 1 lM of the agonist 5-HT reduced FSK -stimu-
lated cAMP to 29 ± 8% of control (Fig. 3). 16 lM
CBD reduced FSK-stimulated cAMP to 38 ± 3% of
control. At a concentration of 0.05 lM, the highly
specific 5-HT1aR antagonist NAN-190 reduced the
5-HT effect to 60 ± 7% of control and the CBD effect
to 76 ± 5% of control, providing further evidence
that CBD is acti ng at the ligand- binding site of H5-
0
50
100
150
200
250
Control 5HT (0.1) CBD (16) 5HT/CBD
% Control (Specific [35S]γ-S-GTP Incorp.)
*
**
Fig. 2. Incorporation of [35S]GTPcS by Cannabidiol (CBD) In Membranes Containing the Human 5-HT1a Receptor. Control represents
incorporation in the basal setting (buffer). Concentrations in micromolar are: 5-HT (0.1); CBD (16). Results are expressed relative to basal
incorporation as mean ± SEM with n’s=9–18. *P<0.01, relative to Control;**P<0.01, relative to 5HT. Further experimental details are
found in Experimental Procedure.
Fig. 3. Inhibition of Forskolin (FSK)-Stimulated cAMP by Cannabidiol (CBD), Serotonin (5-HT), and the inhibitor NAN-190 (NAN) in
Whole Cells Transfected With the Human 5-HT1a Receptor. All conditions contain FSK at 30 lM and the phosphodiesterse inhibitor
isobutylmethylxanthine (IBMX) at 100 lM. Other concentrations in micromolar are: 5-HT (1); CBD (16); and NAN (0.05). Results are
expressed as percentage of FSK control as mean ± SEM with n’s = 3–6. *P<0.05, relative to 5-HT; **P<0.01, relative to CBD. Further
experimental details are found in Experimental Procedure.
1040 Russo, Burnett, Hall, and Parker
HT1aR. At the concentration used here (0.05 lM),
NAN-190 does not reduce FSK-stimulated cAMP
levels on its own (data not shown).
Since the 5-HT2aR is another receptor puta-
tively involved in the pathogenesis of migraine
headache, we conducted a limited comparison at
cloned rat 5-HT2aR. At the highest concentration
of CBD tested (32 lM), 50 ± 5% of [3H]Ketans-
erin is displaced from membrane preparations of
the cloned rat 5-HT2aR. The displacement is con-
centration-dependent as lower concentrations of
CBD progressively displace less ketanserin, until at
8 lM CBD, the effect is barely above control level.
Comparatively, then, CBD is less potent in dis-
placement from the rat 5-HT2aR relative to H5-
HT1aR. As with H5-HT1aR, THC (32 lM) is
inactive in displacement from rat 5-HT2aR. Signal
transduction properties of CBD at rat 5-HT2aR
have not been explored yet.
DISCUSSION
There is substantial literat ure to support the idea
that tetrahydrocannabinol (THC) is responsible for
many of the meaningful and diverse components of
cannabis’ pharmacological activity (33), but other
available evidence supports important contributions
of CBD and other phytocannabinoids and terpenoids
to its pharmacological activity (34,35). It is well
established that the pharmacology of cannabis
combines therapeutic properties (e.g., benefits on
neuropathic pain and spastici ty) (36–39), and lower
urinary tract symptoms (40) that must be weighed
against adverse effects such as intoxication that may
be counter-productive in a therapeutic sense. A
prominent example of the latter is the hallucinogenic
potential of cannabis demonstrated at higher doses,
especially in certain cultural settings. There is also an
outstanding body of experimental evidence to suggest
that THC is hallucinogenic while the closely related
cannabinoid, cannabidiol (CBD) opposes such
activity (3,41).
In pursuit of those pharmacologic al actions of
cannabis that may underlie some of its medicinally
important possibilities, differentiation between TH C
and CBD at the receptor level may be of significance.
This could be especially so at non-cannabinoid
receptors such as 5-HT recept ors. The results shown
in Fig. 1 establish such a contrast in that CBD shows
micromolar affinity in displacing a known agonist,
[3H]8-OH-DPAT, from the 5-HT1aR ligand-binding
site, THC is inactive in the same concentration
range.
CBD’s 5-HT1aR potency could underlie activity
anywhere along the intrinsic activity continuum from
full agonist to silent antagonist. Experiments sum-
marized in Figs. 2 and 3 provide evidence that CBD
is likely to behave a s an agonist in this receptor sys-
tem. Thus, CBD demonstrated the ability to increase
GTP binding to the receptor coupled G protein, Gi,
which is characteristic behaviour of a receptor ago-
nist. These GPCR are further linked to effector signal
transduction sub-systems such as, in the case of a Gi
GPCR, the AC step in cAMP regulation. In Fig. 3,
when AC is stimulated by forskolin (FSK), the ago-
nist 5-HT markedly reduces cAMP production in this
negatively coupled complex. Likewise, CBD acts as
an agonist in these experiments by reducing cAMP
concentration. The results in Figs. 2 and 3 together
support the hypothesis that CBD is an agonist.
Although not completely conclusive in demonstrating
whether CBD is a full or partial agonist, the com-
parable power of CBD and 5-HT at concentrations
that represent less than full receptor occupancy
(Fig. 1) lend support to the full agonist concept.
The contrast between CBD and THC regarding
their interactions at 5-HT1aR relative to CB1R is
striking. THC is at least 10 times more potent in
binding to CB1R; at 5-HT1aR the relationship is just
the opposite, where CBD has micromolar affinity,
and THC shows no binding in the micromo lar range.
At CB1R, THC has sub-micromolar affinity, yet
CBD has micr omolar affinity. The comparison con-
tinues into the realm of signal transduction, where at
CB1R, CBD is putatively an antagonist or inverse
agonist (2); at 5-HT1aR, we have concluded that
CBD is an agonist.
What implications do these results at 5-HT1aR
have for CBD and cannabis? Cannabis is a very
complex mixture of chemical compounds (42), as is
true of most crude natural product drug mixtures.
The dearth of biochemical investigations with non-
psychoactive cannabis components, such as CBD,
create a void of understanding regarding the use of
one or more of these pharmacologically active com-
ponents as therapeutic agents. It has recently been
demonstrated that CBD stimulates TRPV1 (one of
the vanilloid receptors), inhibits the reuptake of
anandamide, and weakly inhibits its hydrolysis (42),
thus making it possibly the first pharmacotherapeutic
agent to modulate endocannabinoid function (1). As
anandamide has already shown activity at 5-HT1aR,
and 36% inhibition of function at 5-HT2aR (14), the
5-HT1a Receptor Agon ism by Cannabidiol 1041
psychopharmacological importance of such relation-
ships is underscored.
The results reported here argue that CBD is active
as an agonist in vitro at H5-HT1a R and that CBD may
also have in vitro actions at the rat 5-HT2aR. Should
CBD prove to have antagonistic activity at 5-HT2A, it
would support its role as a migraine prophylactic agent
(19). Together, these results lend credence to the idea
that CBD and related compounds merit study at a
variety of receptor systems, in a number of species, and
at various levels from the molecular to whole animal.
If, for example, CBD demonstrates clinical activity at
5-HT1aR in vivo, therapeutic possibilities could arise
in a variety of neurological and other physiologically
relevant settings.
ACKNOWLEDGMENTS
The authors would like to thank the following individuals for
their assistance and ideas that contributed to this project: Rustem
Medora, Alicia Christians, Cortney Halley, Sonja Sakaske, Ben
Seaver, and Lynn Parker . The following agencies are gratefully
acknowledged for their financial support of the work: NIH NIG-
MS grants #: GM/OD 54302–01 and 02 and NIH NCRR grant #:
P20 RR 15583 to the NIH COBRE Center for Structural and
Functional Neuroscience.
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5-HT1a Receptor Agon ism by Cannabidiol 1043
... The mechanisms responsible for most CBD effects are still not completely elucidated. CBD acts in multiple targets and receptors, such as the serotoninergic system, by activating 5-HT1A receptors 24,31 , and the endocannabinoid system. Essentially, this is a neuromodulator system, which will allow or ceases the neurotransmissions throughout the organism 32 . ...
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Cannabidiol (CBD) is a substance derived from Cannabis sativa, widely studied in medicine for controlling neural diseases in humans. Besides the positive effects on humans, it also presents anxiolytic proprieties and decreases aggressiveness and stress in mammals. Therefore, CBD has the potential to increase welfare in reared animals, as it seems to reduce negative states commonly experienced in artificial environments. Here, we tested the effect of different CBD doses (0,1,10, and 20 mg/kg) on aggressiveness, stress, and reproductive development of the Nile tilapia ( Oreochromis niloticus ) a worldwide fish reared for farming and research purposes. CBD mixed with fish food was offered to isolated fish for 5 weeks. The 10 mg/kg dose decreased fish’s aggressiveness over time, whereas 20 mg/kg attenuated non-social stress. Both doses decreased the baseline cortisol level of fish and increased the gonadosomatic index. However, CBD 1 and 10 mg/kg doses decreased the spermatozoa number. All CBD doses did not affect feeding ingestion and growth variables, showing that it is not harmful to meat production amount. Despite the effect on spermatozoa, CBD supplementation exhibits high potential to benefit animals’ lives on an integrative-based welfare approach. Therefore, we showed for the first time that CBD could be used as a tool to increase non-mammal welfare, presenting a great potential to be explored in other husbandry and captivity species.
... Furthermore, CBD exerts agonistic activity at the serotonin receptor 5-HT1A (Russo et al., 2005) and acts as an allosteric inhibitor at 5-HT3A receptors (Yang et al., 2010) widely distributed in the body. CBD induces various 5-HT1A-mediated physiological responses (Turner et al., 2017;de Almeida and Devi, 2020). ...
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The European Commission has determined that cannabidiol (CBD) can be considered as a novel food (NF), and currently, 19 applications are under assessment at EFSA. While assessing these, it has become clear that there are knowledge gaps that need to be addressed before a conclusion on the safety of CBD can be reached. Consequently, EFSA has issued this statement, summarising the state of knowledge on the safety of CBD consumption and highlighting areas where more data are needed. Literature searches for both animal and human studies have been conducted to identify safety concerns. Many human studies have been carried out with Epidyolex®, a CBD drug authorised to treat refractory epilepsies. In the context of medical conditions, adverse effects are tolerated if the benefit outweighs the adverse effect. This is, however, not acceptable when considering CBD as a NF. Furthermore, most of the human data referred to in the CBD applications investigated the efficacy of Epidyolex (or CBD) at therapeutic doses. No NOAEL could be identified from these studies. Given the complexity and importance of CBD receptors and pathways, interactions need to be taken into account when considering CBD as a NF. The effects on drug metabolism need to be clarified. Toxicokinetics in different matrices, the half-life and accumulation need to be examined. The effect of CBD on liver, gastrointestinal tract, endocrine system, nervous system and on psychological function needs to be clarified. Studies in animals show significant reproductive toxicity, and the extent to which this occurs in humans generally and in women of child-bearing age specifically needs to be assessed. Considering the significant uncertainties and data gaps, the Panel concludes that the safety of CBD as a NF cannot currently be established.
... It is worth mentioning that CBD, besides modulating the receptors cited above, is also capable of modulating 5-HT 1A , PPAR, α1β, and α1 glycine receptors and transient receptor potential channel of ankyrin type-1 (TRPA1) (Russo et al., 2005;Ahrens et al., 2009;De Petrocellis et al., 2011). This broad spectrum of action gives this molecule the potential to be used in a wide range of physiopathological events, which have been more intensely explored in the last years. ...
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Historically, Cannabis is one of the first plants to be domesticated and used in medicine, though only in the last years the amount of Cannabis-based products or medicines has increased worldwide. Previous preclinical studies and few published clinical trials have demonstrated the efficacy and safety of Cannabis-based medicines in humans. Indeed, Cannabis-related medicines are used to treat multiple pathological conditions, including neurodegenerative disorders. In clinical practice, Cannabis products have already been introduced to treatment regimens of Alzheimer’s disease, Parkinson’s disease and Multiple Sclerosis’s patients, and the mechanisms of action behind the reported improvement in the clinical outcome and disease progression are associated with their anti-inflammatory, immunosuppressive, antioxidant, and neuroprotective properties, due to the modulation of the endocannabinoid system. In this review, we describe the role played by the endocannabinoid system in the physiopathology of Alzheimer, Parkinson, and Multiple Sclerosis, mainly at the neuroimmunological level. We also discuss the evidence for the correlation between phytocannabinoids and their therapeutic effects in these disorders, thus describing the main clinical studies carried out so far on the therapeutic performance of Cannabis-based medicines.
... Unlike THC, CBD has no hedonic effects (10). Based on the current understanding of CBD, it appears to be a modulator of the endocannabinoid system [a weak antagonist of CB1; (11)], a serotonin receptor agonist via the 5-HT1a receptors (12)(13)(14) and an allosteric modulator of the µ and δ opioid receptors (15). It may also have an impact on the glutamatergic system (12). ...
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Introduction: Cannabidiol (CBD), the second most prevalent cannabinoid found in cannabis, is considered to be safe for use. Studies suggest that CBD may be of benefit in treating cannabis use disorder (CUD). In clinical practice, CBD is already being used by patients who are trying to reduce or stop their cannabis consumption. The aim of this study was to assess the potential of CBD inhaled using a vaping device in CUD. Methods: This was an exploratory, observational, non-randomized, open-label study conducted at an Addiction Support and Prevention Center in Paris. The primary endpoint was a reduction of at least 50% in the reported number of joints consumed daily at 12 weeks. The participants were given an electronic cigarette along with liquid containing CBD. Nicotine at 6 mg/ml could be added in case of co-consumption of tobacco. They were assessed once a week and the CBD liquid dose was adjusted based on withdrawal signs and cravings (33.3, 66.6 or 100 mg/mL). Results: Between November 2020 and May 2021, 20 patients were included and 9 (45%) completed the follow-up. All of the participants used tobacco, and were provided a liquid with nicotine. At 12 weeks, 6 patients (30%) had reduced their daily cannabis consumption by at least 50%. The mean number of joints per day was 3, compared to 6.7 at baseline. The mean amount of CBD inhaled per day was 215.8 mg. No symptomatic treatment for cannabis withdrawal was prescribed. Mild adverse effects attributable to CBD and not requiring the prescription of any medicines were reported in a few patients. Conclusion: This research provides evidence in favor of the use of CBD in CUD. It also highlights the benefits of inhalation as the route of CBD administration in patients who use cannabis: inhalation can allow users to self-titrate CBD based on their withdrawal symptoms and cravings. This study illustrates the interest of proposing an addictological intervention targeting at the same time tobacco and cannabis dependence in users who are co-consumers. A double-blind, randomized, placebo-controlled clinical trial is needed to assess the efficacy of inhaled CBD in CUD.Study registration number (IDRCB) issued by the ANSM (Agence nationale de sécurité du médicament et des produits de santé-French National Agency for Medicines and Health Products Safety): 2018-A03256-49. This study received IEC approval from the CPP Sud-Ouest et Outre-Mer 1 (South-West and Overseas 1 IEC) on 15/06/2020 (CPP 1-19-041/ID 3012).
... The main ones include Δ 9 -tetrahydrocannabinol (THC), which serves as a CB1R and CB2R partial agonist [15], as well as cannabidiol (CBD; Figure 1A), which serves as a CB1R negative allosteric modulator [16]. Its additional mechanisms include but are not limited to modulation of 5-HT1A [17] and GPR55 [18,19]. Each of these two molecules has been reported to affect various mechanisms involving obesity pathophysiology. ...
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Obesity is a global medical problem; its common form is known as diet-induced obesity (DIO); however, there are several rare genetic disorders, such as Prader–Willi syndrome (PWS), that are also associated with obesity (genetic-induced obesity, GIO). The currently available therapeutics for treating DIO and GIO are very limited, and they result in only a partial improvement. Cannabidiolic acid (CBDA), a constituent of Cannabis sativa, gradually decarboxylates to cannabidiol (CBD). Whereas the anti-obesity properties of CBD have been reasonably identified, our knowledge of the pharmacology of CBDA is more limited due to its instability. To stabilize CBDA, a new derivative, CBDA-O-methyl ester (HU-580, EPM301), was synthesized. The therapeutic potential of EPM301 in appetite reduction, weight loss, and metabolic improvements in DIO and GIO was tested in vivo. EPM301 (40 mg/kg/d, i.p.) successfully resulted in weight loss, increased ambulation, as well as improved glycemic and lipid profiles in DIO mice. Additionally, EPM301 ameliorated DIO-induced hepatic dysfunction and steatosis. Importantly, EPM301 (20 and 40 mg/kg/d, i.p.) effectively reduced body weight and hyperphagia in a high-fat diet-fed Magel2null mouse model for PWS. In addition, when given to standard-diet-fed Magel2null mice as a preventive treatment, EPM301 completely inhibited weight gain and adiposity. Lastly, EPM301 increased the oxidation of different nutrients in each strain. All together, EPM301 ameliorated obesity and its metabolic abnormalities in both DIO and GIO. These results support the idea to further promote this synthetic CBDA derivative toward clinical evaluation in humans.
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Background: Cannabis-based formulations are now widely used by patients with neurological and psychiatric problems but no studies have been published on the clinical utility of CBD/CBG enriched extracts for parkinsonism's symptoms. Objectives: To describe preliminary clinical data collection of PD and DLB patients under CBD/CBG medical prescription. Methods: Review of electronic records of 14 PD and 5 DLB patients. Four extracts were available 1) CBD broad spectrum (100 mg/ mL) 2) CBD/CBG broad spectrum (100 mg/mL 2:1) (3) CBD/CBG (2:1) + THC0.3% full spectrum (100mg/mL) 4) CBD+THC0.3% full spectrum (100 mg/mL). All the patients received authorization from ANVISA (Brazil) to import the formulations for medical use. Outcomes of each unmet need (UMN) were tabulated and graded. Results: Demographics: PD N = 14 (10 male). DLB: N = 5 (3 male). Mean age: PD: 76.2 yrs. (46-94). DLB: 82.2 yrs. (83-92). Disease duration: PD (6.57 yrs.), DLB (4.2 yrs.); PD H/Y stage (3); PD levodopa dose: 490 mg (150-900). Mean daily doses: PD CBD: 65.17 mg (8.33-125 mg), CBG: 22,50 mg (4.16-50 mg), THC: 2,32 mg (0,75-4,5 mg). DLB CBD: 52 mg (5-100 mg). CBG: 8,75 mg (2,5-15 mg), THC: 0,225 mg. Positive results were seen for RBD, insomnia, anxiety, and pain. All pain responders were on CBG and/or THC formulations. Hallucinations were also attenuated in both patient groups. Safety and tolerability were favorable in this small sample. Conclusions: Future clinical trials in Parkinson's disease and DLB with cannabinoids should focus on their potential benefit for associated anxiety, and pain. The potential anti-psychotic effects of CBD and CBD/CBG should also be further evaluated in a phase 2a clinical trial. Abstract Citation: Flávio Henrique de Rezende Costa., et al. "Parkinson's Disease and Dementia with Lewy Bodies, Patients Under Treatment with Standardized
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The latest years have witnessed a growing interest towards the relationship between neuropsychiatric disease in children with autism spectrum disorders (ASD) and severe alterations in gut microbiota composition. In parallel, an increasing literature has focused the attention towards the association between derangement of the endocannabinoids machinery and some mechanisms and symptoms identified in ASD pathophysiology, such as alteration of neural development, immune system dysfunction, defective social interaction and stereotypic behavior. In this narrative review, we put together the vast ground of endocannabinoids and their partnership with gut microbiota, pursuing the hypothesis that the crosstalk between these two complex homeostatic systems (bioactive lipid mediators, receptors, biosynthetic and hydrolytic enzymes and the entire bacterial gut ecosystem, signaling molecules, metabolites and short chain fatty acids) may disclose new ideas and functional connections for the development of synergic treatments combining “gut-therapy,” nutritional intervention and pharmacological approaches. The two separate domains of the literature have been examined looking for all the plausible (and so far known) overlapping points, describing the mutual changes induced by acting either on the endocannabinoid system or on gut bacteria population and their relevance for the understanding of ASD pathophysiology. Both human pathology and symptoms relief in ASD subjects, as well as multiple ASD-like animal models, have been taken into consideration in order to provide evidence of the relevance of the endocannabinoids-microbiota crosstalk in this major neurodevelopmental disorder.
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Chapter
Following injury, the endocannabinoid system is activated in the brain suggesting a strategic role in the self-repair mechanisms. Indeed endocannabinoid system manipulation ameliorates traumatic brain injury (TBI) symptoms. Cannabidiol (CBD), together with △⁹-tetrahydrocannabinol (THC), is the main phytocannabinoid extracted from the plant Cannabis sativa, and it plays anti-inflammatory, antioxidant, neuroprotective, anticonvulsant, hypnotic, and antiemetic effects and has proven to be useful in neuropsychiatric, neurodegenerative, post-traumatic stress, and ischemic disorders. Unlike THC, CBD is not psychoactive and enhances the beneficial and reduces the side effects of THC. CBD has negligible action on cannabinoid receptors and modulates the endocannabinoid system throughout the inhibition of endocannabinoid degradation and reuptake. It also stimulates serotonin 1A (5-HT1A), adenosine 2A (A2A), transient receptor potential vanilloid subtype 1 (TRPV1), and nuclear peroxisome proliferator-activated receptor γ (PPARγ). We collect in this chapter all the preclinical and clinical evidence on the beneficial effects of CBD in the TBI considering it important for two main reasons: the lack of effective therapy for the TBI and the good tolerability of the CBD.
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Cannabis has been employed in human medicine for more than 4000 years. In the last century, political prohibition led to its disappearance from the conventional pharmacopoeia, but this trend is reversing due to the broad acceptance and application of this forbidden medicine by patients with chronic and intractable disorders inadequately treated by available therapeutics. This study addresses the “road back” for cannabis medicines, and reacceptance as prescription products.Current pharmacology of the two primary therapeutic phytocannabinoids, THC and CBD, is reviewed with respect to herbal synergy and as pertains to treatment of pain, spasm and the wide range of therapeutic applications and adverse effects of cannabis.In particular, the efforts of GW Pharmaceuticals to develop cannabis based medicine extracts (CBME) are documented including cultivation of genetically-selected medical-grade cannabis cloned strains in glass houses with organic and integrated pest management techniques, and their processing employing supercritical carbon dioxide extraction and winterization. These CBMEs are then available for formulation of dosage forms including sublingual extracts and inhaled forms. An optional Advanced Delivery System (ADS) is also discussed.
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. A central tenet underlying the use of botanical remedies is that herbs contain many active ingredients. Primary active ingredients may be enhanced by secondary compounds, which act in beneficial syn-ergy. Other herbal constituents may mitigate the side effects of dominant active ingredients. We reviewed the literature concerning medical can-nabis and its primary active ingredient, ∆ 9 -tetrahydrocannabinol (THC). Good evidence shows that secondary compounds in cannabis may enhance the beneficial effects of THC. Other cannabinoid and non-cannabinoid compounds in herbal cannabis or its extracts may reduce THC-induced anxiety, cholinergic deficits, and immunosuppression. Cannabis terpenoids and flavonoids may also increase cerebral blood flow, enhance cortical activity, kill respiratory pathogens, and provide anti-inflammatory activ-ity. [Article copies available for a fee from The Haworth Document Delivery Service: and: Cannabis Therapeutics in HIV/AIDS (ed: Ethan Russo) The Haworth Integrative Healing Press, an imprint of The Haworth Press, Inc., 2001, pp. 103-132. Single or multiple copies of this arti-cle are available for a fee from The Haworth Document Delivery Service [1-800-342-9678, 9:00 a.m. -5:00 p.m. (EST). E-mail address: getinfo@haworthpressinc.com].
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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This chapter discusses measurement of receptor-stimulated guanosine 5'-O-(γ-Thio)triphosphate binding by G Proteins. Many transmembrane signaling processes caused by extracellular hormones and neurotransmitters are mediated by receptors interacting with heterotrimeric (αβγ) guanine nucleotide-binding proteins (G proteins) attached to the inner face of the plasma membrane. Agonist-liganded receptors apparently initiate activation of G proteins by catalyzing the exchange of guanosine 5'-diphosphate (GDP) by guanosine 5'-triphosphate (GTP) bound to the α subunits. In membrane preparations and reconstituted systems, this activation process is frequently monitored by studying agonist stimulation of high-affinity GTPase, an enzymatic activity of G-protein α subunits. To study the initial steps of G-protein activation by agonist-liganded receptors in a quantitative manner, the binding of radiolabeled GTP analogs, which are not hydrolyzed by the GTPase activity of G-protein α subunits, to G proteins is determined. Of these GTP analogs, guanosine 5'-O-(γ-[35S]thio)triphosphate ([35S]GTPγS) is most frequently used. This nucleotide has a high affinity for all types of G proteins and is available with a relatively high specific radioactivity (1000-1400 Ci/mmol; physical half-life 87.4 days). The chapter describe the measurement of receptor induced binding of [35S]GTPγS to membranous and detergent-solubilized G proteins and how this method can be adapted to different G proteins for an optimal response to receptor stimulation.
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(³H)Spiroxatrine was examined as a potential ligand for the labeling of 5-HT/sub 1A/ sites in the rat hippocampus. Analysis o the binding of (³H)spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity for the (³H)spiroxatrine binding sites, consistent with the labeling of 5-HT⁠sites, while dopamine and norepinephrine had very low affinity. Saturation studies of the binding of (³H)spiroxatrine revealed a single population of sites with a K/sub d/ = 2.21 nM. Further pharmacologic characterization with the 5-HT/sub 1A/ ligands 8-hydroxy-2-(di-ni-propylamino)tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with (³H)spiroxatrine labeling 5-HT/sub 1A/ sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of (³H)spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. 21 references, 5 figures, 2 tables.
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Serotonin1A (5-HT1A) receptors are located on both 5-HT cell bodies where they act as inhibitory autoreceptors and at postsynaptic sites where they mediate the effects of 5-HT released from nerve terminals. The sensitivity of 5-HT1A receptors in humans can be measured using the technique of pharmacological challenge. For example, acute administration of a selective -HT1A receptor agonist, such as ipsapirone, decreases body temperature and increases plasma cortisol through activation of pre- and postsynaptic 5-HT1A receptors, respectively. Use of this technique has demonstrated that unmedicated patients with major depression have decreased sensitivity of both pre- and postsynaptic 5-HT1A receptors. Treatment with selective serotonin reuptake inhibitors further down-regulates -HT1A receptor activity. Due to the hypotheses linking decreased sensitivity of 5-HT1A autoreceptors with the onset of antidepressant activity, there is current interest in the therapeutic efficacy of combined treatment with selective serotonin reuptake inhibitors and -HT1A receptor antagonists.
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Serotonin1A (5-HT1A) receptors are located on both 5-HT cell bodies where they act as inhibitory autoreceptors and at postsynaptic sites where they mediate the effects of 5-HT released from nerve terminals. The sensitivity of 5-HT1A receptors in humans can be measured using the technique of pharmacological challenge. For example, acute administration of a selective 5-HT1A receptor agonist, such as ipsapirone, decreases body temperature and increases plasma cortisol through activation of pre- and postsynaptic 5-HT1A receptors, respectively. Use of this technique has demonstrated that unmedicated patients with major depression have decreased sensitivity of both pre- and postsynaptic 5-HT1A receptors. Treatment with selective serotonin reuptake inhibitors further down-regulates 5-HT1A receptor activity. Due to the hypotheses linking decreased sensitivity of 5-HT1A autoreceptors with the onset of antidepressant activity, there is current interest in the therapeutic efficacy of combined treatment with selective serotonin reuptake inhibitors and 5-HT1A receptor antagonists.
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The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.
Article
Migraine is a frequent paroxysmal headache disorder of unknown aetiology. Genetic factors may control attack frequency and possibly attack severity. Serotonin1D (5-HT1Dβ) receptors have a prominent position within the final common pathway of the mechanisms involved in the headache and associated symptoms. Stimulation of these receptors by selective 5-HT1Dβ receptor agonists such as sumatriptan and newer compounds including MK-462 and 311C90, rapidly and fully blocks the symptoms of the headache phase. The efficacy depends on factors such as timing of administration during or before the headache, speed of initial rise of drug plasma levels, and possibly degree of brain penetration. All agonists at S-HT1Dβ receptors share a short duration of action resulting in recurrence of the headache symptoms within 24 h in about one-third of attacks in clinical trials. The risk for headache recurrence seems patient dependent: about 10% of patients treating multiple attacks experience headache recurrence in every treated attack, whereas 40% never experience recurrence. These differences are not related to simple pharmacokinetic differences between patients or drugs. Increasing plasma half-life of the drug will most likely not reduce the risk of recurrence. “Breakthrough of peripheral suppressive effect” with an ongoing “central migraine generator”, rather than the occurrence of a new attack, seems to be the most likely underlying mechanism for headache recurrence. In a minority of, possibly predisposed, patients, use of sumatriptan may induce increase of attack frequency. Four mechanisms have been suggested for the antimigraine action of 5-HT1Dβ receptor agonists: (1) vasoconstriction of cranial, most likely meningeal and dural blood vessels; (2) inhibition of release of vasoactive neuropeptides from perivascular trigeminal nerve terminals within dura mater and meninges; (3) blockade of trigeminal nerve terminal depolarization; and (4) central inhibition within the trigeminal nucleus caudatus in the brainstem. Which of these mechanisms is the most important, and whether or not vasoconstrictor action is necessary for antimigraine efficacy, is currently under extensive investigation. At this point all drugs with proven antimigraine efficacy share the ability to contract blood vessels and thus all feature also the potential risk of causing vasoconstriction of coronary vessels. In relation herewith, major efforts are put into the search for “the antimigraine receptor” and which receptor subtype mediates which action of sumatriptan-like drugs. At this point, the 5-HT1Dβ receptor subtype is thought to mediate vasoconstriction. Some investigators feel that the 5-HT1D receptor subtype mediates the neuronal effects of sumatriptan, while others are much less convinced about the physiological role of this subtype of receptor. Further research into receptor subtype specificity and affinity of compounds may promote the development of even better antimigraine drugs.